Post-translational modifications such as ubiquitination and phosphorylation play an important role in the regulation of cellular protein function. HIPK2 is a member of the recently identified family of nuclear protein kinases (HIPK1, HIPK2 and HIPK3) that act as corepressors for homeodomain transcription factors. HIPK2 localizes to nuclear speckles (dots) by means of the nuclear speckle retention signal (SRS). This speckle retention signal contains a domain that interacts with a mouse ubiquitin-like protein (SUMO-1) conjugating (E2) enzyme, mUBC9. The mUBC9 interaction domain is well conserved among HIPKs. In cultured cells, HIPK2 is covalently modified by SUMO-1 at multiple sites. Indirect immunofluorescence and confocal microscopic analyses of cells cotransfected with myc-HIPK2 and GFP-SUMO-1 revealed that an intensive SUMO-1 signal from GFP fluorescence was detected in the nuclear speckles (dots) in addition to a diffuse signal in the nucleus. Superimposition of the HIPK2 and SUMO-1 signals demonstrates the colocalization of HIPK2 and SUMO-1 in the nuclear speckles (dots). The target lysine residues in HIPK2 for SUMO-1 modification were partially mapped. Mutation of the target lysine residues prevents SUMO- 1 modification and consequently inhibits localization of HIPK2 to nuclear speckles (dots). Furthermore, the unmodified HIPK2 binds to NK- 3 and the Groucho corepressor. Taken together, these results provide the first evidence that the nuclear protein kinase HIPK2 can be covalently modified by SUMO-1, which directs its localization to nuclear speckles (dots) and modulates its activity. - HIPK, protein kinase, homeodomain, transcription regulation, SUMO-1, corepressor